Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer death in American men. The utility of the widely accepted diagnostic test for PCa, the PSA test, has come under severe scrutiny as only 20-30% of men with PCa have levels that fall within the current PSA reference range. Elevated PSA levels are not correlated well with the stage of PCa and a significant number of men are exposed to unnecessary surgery and radiation therapy with concomitant risks and detrimental side effects. Molecular imaging of PCa is an increasingly important component for diagnosis, staging and treatment of the disease. However, the FDA has approved only one diagnostic agent for PCa since 1996, ProstaScint, a mouse monoclonal antibody. While the marker that ProstaScint recognizes, prostate-specific membrane antigen (PSMA), is ideal as a biomarker, ProstaScint binds an intracellular epitope, rendering it unable to detect living PCa cells. As a result, diagnostic accuracy of less than 50% has led to poor adoption of this imaging test by oncologists. PSMA, a transmembrane protein, has been described as an ideal biomarker for PCa diagnosis and targeted therapy as it 1) is expressed mainly in PCa with a prevalence of >90%, 2) has high expression levels at all stages of the disease and is upregulated in androgen-insensitive and metastatic disease, 3) is accessible on the cell surface and not shed into the circulation, and 4) is internalized after ligand binding. To address the unmet need for accurate and quantitative imaging of PCa we will advance our novel, proprietary, low molecular weight PSMA inhibitor that selectively delivers an imaging radionuclide to PCa cells. This small molecule inhibitor, CTT1057, binds PSMA irreversibly at an IC50 of less than 1 nM. In addition, CTT1057 and its 18F imaging payload is internalized into PSMA expressing cells in less than 30 minutes. This irreversible binding and rapid internalization makes this molecule unique amongst PSMA inhibitors that are in development and allows for excellent biodistribution and tumor uptake. In addition, we are pursuing the more sensitive and quantitative diagnostic imaging modality of PET (as opposed to SPECT) using a 18F-labeled radiotracer. In this grant we will advance the development of our PET imaging agent CTT1057 by completing the cGMP manufacture and automated radiolabeling of Phase I clinical material in Aim 1. In Aim 2, we will establish a human safety profile as well as visualize PCa lesions and determine optimal imaging parameters and dosimetry in a Phase 0/I clinical trial in PCa patients. PET imaging using our 18F-labeled PSMA inhibitor CTT1057 will allow for rapid and sensitive assessment of proximal and distal manifestations of PCa, something which is currently unavailable.